Microscopy

Question 1

Describe what a microscope does and name 4 different types of microscope.

Answer 1

A microscope is an instrument which enables you to magnify an object a lot.

• compound light microscope
• laser scanning confocal microscope
• scanning electron microscope
• transmission electron microscope

Question 2

Label a diagram of a light microscope with the names of the component parts

Answer 2

look at textbook

Question 3

name and write the functions of the parts of a compound light microscope

Answer 3

1. Eye piece- is what you look through and it magnifies the object 10x
2. Scanning lens- It is used in preliminary observation and magnifies 4x.
3. Low power objective lens- magnifies 10x and gives the middle amount of detail.
4. High power objective lens- magnifies 40x and gives the most amount of detail.
5. Stage clips- used to secure the slide and hold it in place
6. Stage- stand above the the light source for the glass slide.
7. Arm- where you hold a microscope
8. Condenser- used to vary the intensity of the light projected
9. Coarse- roughly puts things into focus and should only be used with the scanning lens.
10. Fine adjustment- Puts things into full focus and should only be used with the low and high power objectives lenses.
11. Illumination- projects light onto the slide.

Question 4

Outline how an SEM works

Answer 4

A beam of electrons is fired at the surface of a specimen and the reflected electrons are collected to create a 3D image.

Question 5

Outline how a TEM works

Answer 5

A beam of electrons is transmitted through the specimen and focused to produce an image.

Question 6

What is flourescence

Answer 6

It is the absorption and re radiation of light.

Question 7

Outline how a laser scanning lens works

Answer 7

It moves a single spot of focused light across a specimen (point of illumination). this causes fluorescence with the components labelled with a dye.
The emitted light from the specimen is filtered through a pin hole aperture. Only light radiated from very close to the focal plane (the distance that gives the sharpest distance) is detected.

Question 8

Draw a table comparing the use and properties of light, SEM, TEM and laser scanning confocal microscope.

Answer 8

look at book

Question 9

State the features of the images produced from light, SEM, TEM and laser scanning confocal microscopes.

Answer 9

look at book

Question 10

Know a bit about the history…

Answer 10

look at book

Question 11

Explain how to use a light microscope to view a specimen at low and high powers.

Answer 11

1. When using low power lenses ( scanning or low power objective lens) use the coarse to roughly bring it to focus.
2. When using a high power use the fine adjustment knob.

Question 12

Describe how to produce a temporary wet mount of living tissue.

Answer 12

Specimens are suspended in liquid such as water or an immersion oil.
A cover slip is placed on from an angle, aquatic samples and other living organisms can be viewed this way.

Question 13

Describe and explain the characteristics of a good slide preparation.

Answer 13

1. no air bubble- artefact
2. staining if needed
3. glass and transparent
4. coverslip - protection
5. thin specimen- good quality and a good size
6. good liquid levels.
7. clean

Question 14

Explain why slide preparations need to be thin.

Answer 14

1. transmitted light is going through so it needs to be transparent or you won’t see anything
2. you want to look at a single cell so you can measure it which you can’t do if they overlap.

Question 15

Explain how to use a stage micrometer to work out the distance represented by the small divisions in an eyepiece graticule under 3 different objective lenses.

Answer 15

1. Line up the eye piece graticule with the stage micrometer (the fixed scale on the slide).
2. get the scale on the micrometer slide in focus.
3. align the micrometer scale with the scale in the eyepiece take a reading from both of them.
4. See what the equivalent value of stag units is to eyepiece units.
5. work out the stage units in mm by dividing by ten ( 1 stage unit = 0.1 mm)
6. Divide the stage units in mm by the number of eyepiece units.
7. then put this in the right unit (micrometre) by multiplying by 1000 (magnification factor)

Question 16

Explain how to use a stage micrometer and eye-piece graticule to add a scale bar to a drawing.

Answer 16

1. measure the specimen with the eyepiece graticule.
2. then multiply it by the magnification factor
3. this will get the size of the specimen in micrometres
4. draw specimen and put a bar underneath.

Question 17

Explain how to use a stage micrometer and eye-piece graticule to calculate the
size of a specimen.

Answer 17

1. measure the specimen with the eyepiece graticule.
2. then multiply it by the magnification factor
3. this will get the size of the specimen in micrometres

Question 18

Describe how to choose an appropriate number of significant figures, or decimal places to present data.

Answer 18

Use the highest number of decimal places in the question for your answer.

Question 19

Explain how an adjustment to the “plane of focus” can alter what is viewed within a cell.

Answer 19

If you change the focus while looking through a microscope you will notice different parts of the image will come into focus and other parts will go out of focus.
The plane of focus is the horizontal plane where what you are looking at is in focus.
It is usually not very thick so it is hard to focus on things that are not flat.
When you focus the microscope you move the plane of focus up and down, which is why different parts of what you are looking at come in and out of focus.

Question 20

Explain how a tissue slice might be misleading due to the very thin nature of the slice.

Answer 20

1. you can only see a cross-section of a cell not it’s overall shape- you might not see the actual shape of the cell.
2. there are different organelles at different parts of the cell- you might miss structures that are inside the cell.
3. Its not actually the diameter of the cell just the diameter of the cross section.

Question 21

Explain why staining is useful for light microscopy

Answer 21

1. a lot of specimens are transparent and colourless so difficult to see.
2. staining provides colour- contrast with the background
3. differential staining- you can distinguish between two types of organisms or two types of organelles of a single organisms.

Question 22

Describe the properties a stain needs to have to be useful for light microscopy.

Answer 22

1. Bind to other molecules- differential staining - bind to certain parts of the cell
2. liquid- and be able to get through cell membrane.
3. shouldn’t damage the cell
4. very intense colour- to be able to colour something in small amounts.

Question 23

Describe how to prepare a stained specimen for viewing under a light microscope.

Answer 23

1. place sample on a slide an allow it to air dry
2. this is then heat-fixed by passing through a flame.
3. the specimen will adhere to the microscope slide and will take up stains.

Question 24

Name two common stains and the molecules they bind to.

Answer 24

1. methylene blue- DNA
2. Giemsa- used to differentiate between different types of blood cell.
3. Eosin- cytoplasm turn pink

Question 25

State the rules for biological drawings.

Answer 25

1. include a title
2. state magnification
3. use a sharp pencil for drawings and labels
4. use white unlined paper
5. make it big
6. draw smooth continous lines
7. do not shade
8. draw clearly defined structures
9. ensure proportions are correct
10. label lines should not cross and should not have arrow heads
11. label lines should be parallel to the top of the page and drawn with a ruler

Question 26

Produce a labelled and annotated low power tissue plan using a light microscope.

Answer 26

look at book

Question 27

Produce a labelled and annotated high power drawing of a named number of cells using a light microscope.

Answer 27

look at book

Question 28

State the magnification formula and represent the magnification formula in a triangle diagram.

Answer 28

magnification= size of image/ acutal size of object

Question 29

Explain how to calculate the actual size of an image using the magnification formula.

Answer 29

1. Measure it in mm
2. convert to um by multiplying by 1000
3. then divide it by magnification to get actual size of image.

Question 30

State the symbols used for millimetres, micrometres and nanometres.

Answer 30

milimetres- mm
micrometres- um
nanometres- nm

Question 31

Explain how to convert measurements from one unit into another.

Answer 31

mm-> um    x1000
mm-> nm    x 1,000,000
um-> mm    /1000
um-> nm     x1000
nm-> mm    / 1,000,000
nm-> um     / 1,000

Question 32

Define resolution

Answer 32

the shortest distance between the two objects that are still seen as two separate objects

Question 33

Define magnification

Answer 33

how much bigger an image form a microscope is compared to the specimen

Question 34

State the resolution and useful maximum magnification of light microscopes,

Answer 34

resolution- 200nm

magnification- x1500 - x2000

Question 35

State the resolution and useful maximum magnification of SEMs

Answer 35

resolution- 3-10nm

magnification- x100,000-500,000

Question 36

State the resolution and useful maximum magnification of light microscopes, TEM.

Answer 36

resolution- 0,2-0.5nm

magnification- 500,000- x 2,000,000